Dendritic cells (DCs) are the most potent type of antigen-presenting cells controlling both innate and adaptive immune responses. DCs are continually differentiated from hematopoietic progenitors, ensuring that self versus non-self discrimination is maintained in the steady-state and during immunological challenges. However, how DCs are developed at the molecular level remains incompletely understood. MicroRNAs (miRNAs) have emerged as one of the critical regulatory molecules in development, cell proliferation, differentiation and tumorigenesis. Our long-term goal is to identify specific miRNAs and define their roles in DC development and function. The objective of this project is to determine whether specific miRNAs are critical in human monocyte-derived DC differentiation and function. Our hypothesis is that the process of monocyte differentiation into distinct immature and mature DCs and the immunological functions of differentiated cells are regulated by specific miRNAs. In Aim 1, we will identify and validate the target genes regulated by a specific miRNA that is highly up-regulated in both immature and mature DCs. Our preliminary data suggest that this miRNA may play a role in modulating DC apoptosis through a feedback mechanism. To identify the miRNA targets, we will perform an mRNA expression microarray using RNA from immature DCs that are either over- expressing or have this miRNA expression silenced. Expression of mRNA will be profiled in both experiments, and the intersected mRNA that are decreased and increased will be identified. Molecular validation assays (a luciferase reporter assay, qPCR, Northern and Western blot) and DC apoptosis analysis after knocking down or over-expressing the candidate gene will be carried out. In Aim 2, we will evaluate the impact of altered expression of three specific miRNAs on human DC development and function in vitro. Our preliminary data demonstrate that expression of these miRNAs is significantly altered during DC development. To address this question, silencing or over-expressing these miRNAs will be performed in monocytes and DCs. The effects of altering these miRNA expression levels on DC development, maturation and physiological functions will be analyzed using both molecular and immunological assays. The proposed research is innovative and significant because the role of miRNA in DC development and function remains unexplored. New target genes that are differentially regulated by miRNAs during DC development will be identified. Our work also has translational relevance, as there is a great deal of interest in developing clinical applications using miRNAs to modulate DC development and functions in a broad spectrum of diseases including infections, autoimmunity, cancer, transplantation-related graft versus host disease, and graft rejection PUBLIC HEALTH RELEVANCE: Our research will identify novel small RNA molecules that regulate the development and function of human dendritic cells, the most potent type of white blood cells that keep the immune system in balance. Our research may lead to the development of new small molecules to modulate dendritic cell development and function in infection, allergy, cancer, autoimmunity, and transplantation.